The retinal pigment epithelium (RPE) plays a pivotal role in the development and function of the outer retina. We are interested in RPE-specific mechanisms, at both the regulatory and functional levels, and we have been studying the function and regulation of RPE65, a gene whose expression is restricted to the RPE, and mutations in which cause severe blindness in humans. The phenotype of the Rpe65 knockout mouse is due to disruption of the RPE-based vitamin A visual cycle whose role is to regenerate visual pigment chromophore by isomerization of vitamin A. We have shown that in the Rpe65 knockout mouse there is overaccumulation of all-trans-retinyl esters and total absence of 11-cis-retinal, suggesting a role related to that of retinol isomerohydrolase, the crucial enzyme in visual pigment chromophore regeneration. We have established a catalytic role for RPE65 in the synthesis of 11-cis retinol, identifying it as the long-sought visual cycle isomerohydrolase. We have also continued studies on beta-carotene 15,15'-monooxygenase (BCMO1). BCMO1 is closely related to RPE65 and both are members of a newly emerging diverse family of carotenoid-cleavage enzymes. We postulate that BCMO1 and RPE65 share a similar mechanism of action. Because they share structural features, including identical residues in the catalytic assemblage, we have found BCMO1 to be a useful model for our mechanistic studies addressing RPE65.[unreadable] [unreadable] In the past year we have made the following progress: [unreadable] [unreadable] a) While half of human RPE65 mutations are missense mutations, and many of these do not completely abolish isomerase activity (hypomorphic mutations), we do not have a clear understanding of why residual (10%) RPE65 activity allows usable vision early in life, while delaying but not preventing eventual retinal degeneration. We investigated the effect of missense mutations in RPE65 associated with less severe degenerations to understand their effects on protein structure and stability. We have found that many of these mutants have low isomerase activity (5-10%), but their carriers can retain usable vision into 3rd or 4th decade of life. In a collaboration with German clinicians who identified a P25L mutation in a young patient, we have expanded the clinical spectrum of RPE65 dystrophy phenotype. It was found that this young patient has near normal visual acuity, combining abolished rod function and night-blindness with functional cone vision. The activity of RPE65/P25L was measured at 8% of wildtype activity. The relative preservation of cone function strongly suggests that human cones can selectively sequester enough 11-cis retinal to maintain their function better than rods. The ultimate goal of this aspect of our research is to determine if the activity of such hypomorphic RPE65 mutants can be augmented by modulation of their stability by pharmacologic agents. Also, in light of recent advances in RPE65 gene therapy, such studies provide a rational assessment of suitability for gene therapy. [unreadable] [unreadable] b) To complement the work reported in (a), we are generating a panel of hypomorphic knock-in mice in the mouse Rpe65 gene by homologous recombination. It is anticipated that these will provide important insight into the variability of RPE65-deficient phenotypes, in comparison with the extreme case of the knockout. In particular, they are anticipated to provide insight into the slower progression of the retinal degeneration such as seen in less severe cases of human RPE65 mutations. They also will provide animal models to test pharmacologic strategies developed in (a). The first of these constructs has been found to be germline and the knock-in line has been established. The second has successfully undergone homologous recombination, and is awaiting injection into embryos. The targeting vector for the third is currently in queue for electroporation into ES cells for homologous recombination.[unreadable] [unreadable] c) Previously, we demonstrated a crucial role in enzymatic activity for histidine and acidic residues in BCMO1 (and RPE65) that we hypothesized to be involved in metal coordination. These observations, in conjunction with the predicted structure of a related bacterial enzyme, Synechocystis apocarotenal oxygenase (ACO) confirmed a catalytic role for iron in this family of proteins, but other crucial aspects of the mechanism (substrate binding, electron transfer, etc.) remained unclear. In light of the ACO structure we are investigating how BCMO1, BCMO2 and RPE65 have evolved to fulfill their functions utilizing the basic structure of the carotenoid oxygenase family. All 3 mammalian members of the family are expressed in retina and/or RPE, but RPE65 is specific to the RPE. In the past year, we conducted experiments to better elucidate the mechanism of BCMO1 by determining which residues in the substrate binding tunnel are necessary for catalytic and substrate binding activity, and to explore the possibility that a carbocation intermediate is formed during the cleavage reaction, as seen for many isoprenoid biosynthesis enzymes. We replaced conserved and substrate tunnel tyrosines and acidic residues by site-directed mutagenesis. We showed that while certain tyrosine residues, per se, are required for activity, conservation of aromaticity at others is sufficient. Our results are consistent with the formation of a substrate carbocation intermediate and cation-pi stabilization of this by the aromatic residues in the substrate binding tunnel. We have built an ab initio model of BCMO1 with the mounted &#946;-carotene and demonstrated that distances between residues crucial for activity and the substrate are less than 5 angstroms, which is consistent with a mechanism involving cation-pi stabilization. In addition, these insights may be valuable towards understanding the mechanism of RPE65.